This invention relates to an assay or detection method which employs an enzyme linked detection endpoint signaling system for the detection and measurement of compounds of interest in assay systems employing ligand binding techniques.
Such ligand binding techniques depend upon the facility inherent in biological molecules, such as receptors, antibodies and nucleic acids, to bind with a high degree of specificity to their respective analogous partner ligand. Owing to this specificity, such techniques have found widespread application in the detection and measurement of many entities ranging from simple chemicals to complex biological molecules, including peptides, proteins, carbohydrates and nucleic acids. Consequently, the technique of ligand binding has become one of the most important tools for biological research and diagnostic assays.
In such ligand binding systems, the specific binding reaction occurs when the ligand is presented to the ligand partner compound. Examples include the antibody-antigen reaction, and the hybridization of complementary nucleic acid sequences. A key feature inherent to all ligand binding assay systems is that, in order to monitor the progress of such binding and thus to obtain a qualitative and/or quantitative indication of the degree of such binding, it is necessary to label, either directly or indirectly, at least one of the ligand partners participating in the ligand binding reaction. This labeled ligand can then be employed to generate a measurable signal by which the reaction is monitored. The relative quantity of signal generated by the labeled ligand will be proportional to the quantity of labeled ligand present and thus can serve to indicate the concentration of the labeled ligand. Examples of such signal generators used include radioactive nuclides (125I, 3H, 14C, 32P, etc.), chemiluminescent or fluorescent compounds (acridinium esters, lanthanide chelates) and enzymes (peroxidase, phosphatase).
Numerous non-radioactive methods have been developed to avoid the hazards, and inconvenience and disposal problems posed by radioactive materials. Examples of non-radioactive labels include: (1) enzymes that catalyze conversion of a chromogenic substrate to an insoluble, colored product (e.g. catalase, horseradish peroxidase, and the like) or catalyze a reaction that yields a fluorescent or luminescent product, and (2) direct fluorescent labels (e.g. fluorescein, isothiocyanate, rhodamine, Cascade blue), which absorb electromagnetic energy in a particular absorption wavelength spectrum and subsequently emit visible light at one or more longer (i.e., less energetic) wavelengths.
In the case of enzymes as primary signal generators, the action of the enzyme (e.g., peroxidase) on an appropriate substrate may itself lead to the generation of a secondary signal which is, for example, chemiluminescent or fluorescent in nature. In this situation, the role of the labeling enzyme is either direct, (i.e., to convert the substrate itself from an inactive to an active, and therefore, detectable compound) or indirect (i.e., to convert the substrate from an inactive to active substance, which is itself an initiator or co-factor in the conversion of an inactive to active compound). An example of the direct substrate is the direct action of alkaline phosphatase on stable dioxetanes, where removal of a phosphate group renders the dioxetanes unstable with a consequent release of quantifiable luminescence. An example of an indirect substrate is the indirect action of peroxidase on luminol, where luminescence is generated from enzyme-catalyzed production of an active oxygen species by breakdown of the peroxide (e.g., H2O2) substrate.
Of those systems which have found the most widespread application, the signal intensity can be low thus limiting the scope of application; in order to overcome this problem, such systems preferably use enhancers (e.g., para-iodo-phenol) or amplifiers (e.g., fluorescent polymers) to increase the complexity of these signal generating methods.
Methods based on enzyme-linked analytes offer the best sensitivity, as a single enzyme molecule typically has a persistent capacity to catalyze the transformation of a chromogenic or luminescent substrate into detectable product. With appropriate conditions and incubation time, a single enzyme molecule can produce a large amount of product and hence yield considerable signal amplification.
Chemiluminescent compounds which have been used in these assays include aminophthalhydrazides, acridans, acridinium esters and dioxetanes. U.S. Pat. No. 5,593,845 discloses, for example, chemiluminescent N-alkylacridancarboxylate derivatives which allow the production of light from the acridan by reaction with a peroxide and a peroxidase. U.S. Pat. No. 5,670,644, discloses improved acridan compounds which, upon reaction with a peroxidase enzyme and a peroxide compound, are converted into a more persistent, intermediate acridinium compound, wherein the center ring is aromatic, which subsequently undergoes a rapid chemiluminescent reaction when the pH is raised.
Additional compounds typically used with peroxidase include, but are not limited to 3,3′,5,5′-tetramethylbenzidine (TMB), luminol, 2,2′-azinodi(3-ethyl benathiazoline sulfonic acid) (ABTS), 3′3-diaminobenzidine-(DAB), and 3-amino-9-ethylcarbazole (AEC).
Among the enzymes used in enzyme-linked detection methods such as immunoassays, detection of oligonucleotides and nucleic acid hybridization techniques, the most commonly used is horseradish peroxidase (HRP). Amino-substituted cyclic phthalhydrazides (e.g., luminol and isoluminol) react with H2O2 and a peroxidase enzyme catalyst (e.g., horseradish peroxidase) under basic conditions to emit of light. This reaction has been used as the basis for analytical methods for the detection of H2O2 and for detecting-peroxidase. One problem with the system is that H2O2 is unstable in conditions of a pH greater than 7.0. However, the reaction preferably occur under alkaline conditions. Consequently, methods and compositions are needed which enhance H2O2 stability and/or activity at a pH greater than about 7.0, but which do not quench or otherwise inhibit the reaction.
Traditionally, compositions such as HF and other acids have been used to quench the reaction. The presence of fluoride in these reactions was considered to be responsible for terminating the reaction which produces a colorimetric, a luminescent, or a fluorescent label. Consequently, reagents comprising fluoride were not considered as suitable for use in these reactions.
In unrelated technology, colloidal stannic oxides, which are generated from Sn(II) and Sn(IV) chlorides, were reportedly used to stabilize phosphorus-free and boron-free cleaning compositions solutions which contained hydrogen peroxide (U.S. Pat. No. 5,736,497).
In the area of dental products, stannous chloride has been used to stabilize gels comprising hydrogen peroxide, sodium fluoride and a bicarbonate (U.S. Pat. No. 5,217,710).
U.S. Pat. No. 5,783,382 discloses methods for stably storing an indicator and an enzyme under sealed conditions using disoxidants, such as tin (II) salts. This is distinguishable from the instant invention which teaches stabilization of the substrate, hydrogen peroxide (H2O2).